## Testing The Concept Of Voltagedependent Na Permeability The Voltageclamp Technique

So far we have just a theory. The critical hypothesis is that Na+ permeability is regulated by the membrane potential. The simple way of testing this hypothesis is to depolarize the cell to various levels and measure the corresponding Na+ permeability. The problem, however, is that as soon as the cell is depolarized, Na+ permeability changes, an action potential is initiated, and because of practical reasons, there is insufficient time to measure the permeability change. This was a major obstacle in the further analysis of the ionic mechanisms that govern the action potential.

The major breakthrough came when Hodgkin and Huxley used an electronic feedback device known as a voltage-clamp amplifier to hold the membrane potential at various levels for indefinite periods of time (Fig. 10).

The voltage-clamp amplifier takes the difference between the actual recorded membrane potential and the desired level and generates sufficient hyperpolarizing or depolarizing current to minimize the difference. The amount of current necessary to hold the membrane potential fixed at the desired level is proportional to the membrane permeability, or conductance, at that particular voltage-clamp level. For example, by measuring the ionic current as a function of time, l(t), and knowing the potential difference (which is constant), the conductance as a function of time, G(t), can be determined simply by using Ohm's law (conductance for our purpose can simply be considered an electrical measurement of permeability, so we will use permeability and conductance interchangeably):

By changing the potential difference with the voltage-clamp amplifier, the corresponding conductances at a variety of different potentials can be determined.

Figure 11 illustrates some typical results. The procedure is as follows. Initially, the membrane potential is at its resting level of —60 mV. It is then artificially changed from the resting level to a new depolarized level (e.g., —35 mV) and held there for 5 msec or longer. The membrane potential is then returned back to its resting level, and the membrane is stepped or clamped to a new depolarized level of —20 mV. By performing a sequence of these voltage-clamp measurements, changes in Na+ permeability as a function of both voltage and time can be determined.

In the upper part of Fig. 11, the horizontal axis shows the time and the vertical axis shows the measured Na+ conductance. As the membrane potential is forced to various depolarized levels from the resting level, there is a graded increase in Na+ permeability. The greater the

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